Introduction

The neuropeptide kisspeptin (encoded by KISS1) and its receptor, KISS1R (formerly known as GPR54), are key regulators of reproduction. Humans and mice with mutations in these genes show impaired puberty, hypogonadism, and infertility (1–3). Kisspeptin activates the reproductive axis by directly stimulating, via KISS1R, gonadotropin-releasing hormone (GnRH) neurons (4). Although kisspeptin is expressed in discrete brain regions (5), it is also present in some peripheral tissues (6–8). Likewise, Kiss1r is also expressed in multiple non-GnRH brain areas and in several peripheral tissues (8–10), including metabolic tissues like fat, liver, and pancreas. This suggests that kisspeptin has additional uncharacterized roles outside of reproduction. Yet, thus far, virtually all research on kisspeptin signaling has focused on reproductive regulation.

Changes in energy status or metabolic signals affect both reproduction and hypothalamic kisspeptin levels (11, 12), which suggests that kisspeptin neurons mediate metabolic effects on reproductive status. However, whether kisspeptin signaling also plays a reciprocal role in regulating energy and metabolic status is unclear. Young Kiss1 KO, Kiss1r KO, and WT mice display no genotype differences in BW (3); however, in that study, BW was only measured before full maturity, and other metabolic parameters were not assessed. Initial studies in male rats found no effects of central kisspeptin on food intake (13, 14), but neither females nor peripheral treatments were examined. In male mice, kisspeptin was recently reported to moderately modify satiety (15) and also alter firing properties of hypothalamic pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) neurons, which kisspeptin-immunoreactive fibers appose (16, 17). Thus, whether kisspeptin also regulates energy balance or metabolism, in addition to governing fertility, remains unresolved. We therefore examined the energetic, metabolic, and diabetic phenotype of adult Kiss1r KO mice, which lack functional kisspeptin signaling, on both standard chow diet and high-fat diet (HFD). Our findings indicate that, besides stimulating the reproductive axis, the kisspeptin system is also an important player in BW, energy balance, locomotion, and glucose regulation.

Results and Discussion

To elucidate potential metabolic roles for kisspeptin signaling, we studied Kiss1r KO mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI71075DS1), an established model of impaired kisspeptin signaling (1, 3, 18, 19). Kiss1r KO mice and WT and heterozygous (Het) littermate controls of both sexes were weighed weekly beginning at week 4. Female Kiss1r KO mice showed normal BW until 8–10 weeks of age, after which they weighed significantly more than WT and Het female littermates (Figure 1A). By 18 weeks, Kiss1r KO females weighed a dramatic 30% more than WT females (P < 0.05; Figure 1A). BW of Het and WT females did not differ. In contrast to the obesity in Kiss1r KO females, BW of adult Kiss1r KO males was similar to that of WT and Het littermates (Figure 1D).

The underlying mechanisms of the obesity and glucose impairment in Kiss1r KO mice is unknown and could reflect impaired kisspeptin signaling in the brain and/or periphery (Kiss1r and kisspeptin are expressed in both areas). In the brain, kisspeptin neurons innervate some anorexigenic POMC and orexigenic NPY neurons (16). Our findings of obesity and reduced energy expenditure in Kiss1r KO mice matches the reported ability of kisspeptin to inhibit NPY and activate POMC cells in situ (17). However, the reduced (rather than increased) feeding in our obese Kiss1r KO mice argues against this simple explanation of dysregulated NPY and POMC and suggests that multiple pathways might be affected. Alternatively, or concurrently, the metabolic and diabetic phenotypes in Kiss1r KO mice may arise due to peripheral absence of kisspeptin signaling, as KISS1R is found in adipose, liver, stomach, and pancreas. In vitro and in vivo kisspeptin treatment augments glucose-induced insulin secretion (7, 22), supporting a possible role in pancreatic β cell function, which could potentially underlie the impaired glucose tolerance we observed in Kiss1r KO females. If so, whether such pancreatic signaling would also alter energy balance, adiposity, feeding, or locomotion is unknown. Indeed, different phenotypic parameters in Kiss1r KO mice (e.g., adiposity, locomotion, and glucose homeostasis) may independently reflect impaired kisspeptin signaling in different target pathways/tissues. Moreover, whether the diabetic or metabolic phenotypes occur independently, or as indirect or secondary consequences of the obesity, remains to be determined. Finally, the cause of the observed sexually dimorphic metabolic phenotype is unclear, especially since both male and female Kiss1r KO mice were equally hypogonadal, and sex differences in Kiss1r expression have not been reported.

The few studies on patients with KISS1 and KISS1R mutations have not yet reported an obesity phenotype (1, 2, 23–25). However, it is difficult to conclude anything yet because these studies were generally performed in young patients, before one might expect adulthood obesity to emerge (based on our mouse data), and body composition, metabolism, and glucose tolerance were not assessed. Furthermore, most patients underwent hormone replacement therapy, which would influence BW and metabolism.

In summary, we showed that, besides governing reproduction, kisspeptin signaling is also an important and novel regulator of BW, adiposity, metabolism, and glucose homeostasis, especially in adult females. This newly discovered role for the kisspeptin system extends our understanding of the relationship between reproduction and energy balance and may provide novel insight into various metabolic diseases, such as diabetes, polycystic ovary syndrome, or obesity.

Methods

Further information can be found in Supplemental Methods.

Animals.Kiss1r Het breeders generated Kiss1r KO mice and littermate controls that were genotyped and sexed by PCR of tail DNA. Weaned littermates (∼3 weeks old) were housed at 2–3 per cage (mixed genotype) in a 12-hour light/12-hour dark cycle with ad libitum water and standard rodent chow (3.5 kcal/g, 45.2% available carbohydrate, 11.4% fat, 17.2% crude protein). In one experiment, mice were challenged with HFD (23% fat; 46% of total energy from lipids) for 12 weeks. In some experiments, as noted, mice were GDX under isoflurane anesthetization.

Somatic, endocrine, and metabolic analyses. See Supplemental Methods for details. Briefly, male and female Kiss1r KO mice and control littermates were weighed once or twice weekly. Adult body composition was determined by DEXA. Blood hormone levels were assayed with ELISA kits (leptin) or via RIA (T4; National Hormone and Pituitary Program). i.p. GTT was performed in fasted adult mice. For metabolic and locomotor analyses, indirect calorimetry was performed using either a 12-cage equal-flow CLAMS calorimeter or an 8-cage TSE System calorimeter.

Statistics. All data are mean ± SEM. For data at single points (non–repeated measures), single comparisons were made using 1- or 2-tailed t tests, as appropriate, and multiple comparisons were performed using 1-way ANOVA with Tukey’s post-hoc test. For repeated measures (BW and GTT), 2-way repeated-measures ANOVA was performed, with Bonferroni post-hoc tests directly comparing genotypes at specific points for 3 groups or t tests for 2 groups. A P value less than 0.05 was considered significant.

Study approval. All experiments were approved by UCSD IACUC and Monash University Animal Ethics Committee.